The development of protective or therapeutic HIV vaccines has been hampered by unprecedented challenges, primarily due to the unique properties of the HIV-1 envelope (Env) trimer, a cleverly engineered entry machinery that features an extraordinary assortment of immune-evasion tactics, including antigenic variation, heavy glycosylation of exposed surfaces and conformational camouflage. Further insights into the complex structure-function relationships in the HIV-1 Env trimer and its protective shield will thus be critical to guide the rational design of a protective vaccine. 1) Discovery of a second CD4-binding site (CD4-BS) in the HIV-1 Env trimer. By in silico docking of CD4 to the Env trimer, we found that CD4 makes contact not only with the previously defined CD4-BS in the outer domain of a single gp120 protomer, but also with a second CD4-binding site (CD4-BS2) located in the inner domain of a neighboring protomer. To gain structural insights into the interactions of CD4 with the trimer, we obtained a cryoEM structure of soluble CD4 in complex with a conformationally-constrained soluble trimer, DS-SOSIP.664, which confirmed our model. To elucidate the functional relevance of the quaternary CD4 contact to HIV-1 infection, we mutagenized individual residues in this region in HIV-1 isolates of different genetic subtypes. Strikingly, we found that even single charge inversions of E62/E64, H66 and K207 virtually abrogated the infectivity of all the HIV-1 Env tested, regardless of their genetic clades. These results delineate the initial contact of the HIV-1 Env with the CD4 receptor, documenting the quaternary configuration of the functional HIV-1 receptor-binding site, a feature previously recognized for Picornaviruses, but never hitherto for enveloped viruses. The discovery of a new CD4-binding site paves the way toward the development of new strategies of treatment and vaccine. 2) Optimization of HIV-neutralizing antibodies by introduction of quaternary binding capacity. Broadly neutralizing antibodies (bNAbs) represent a promising alternative to ART for HIV-1 prevention and treatment. We identified a few anti-CD4-BS antibodies, most notably VRC03 and VRC06, which mimic the quaternary-binding mode of CD4 and establish contact with two adjacent gp120 protomers via an extended loop in their heavy chain framework region 3 (HC-FR3). We proved the functional role of this quaternary contact by deleting the FR3 loops of VRC03 and VRC06, which resulted in a near-complete loss of binding and neutralization activity. Since the establishment of quaternary contact appears to bolster the trimer interaction both in CD4 and in selected antibodies, we hypothesized that it might likewise further improve the activity of some of the most potent bNAbs, all of which interact with a single gp120 protomer (e.g., VRC01, VRC07, N6). To validate this assumption, we rationally engrafted the extended FR3 loop of VRC03 onto different CD4-supersite bNAbs and tested the resulting chimeric antibodies against a wide panel of global HIV-1 strains (n = 208). FR3-loop chimerization enhanced the neutralizing activity of potent bNAbs against a majority of global HIV-1 strains. The interactive quaternary surface was delineated by solving the crystal structure of two FR3 loop-chimeric antibodies in complex with a soluble Env trimer. Furthermore, compared to unmodified antibodies, chimeric antibodies displayed a reduced autoreactivity and a prolonged in vivo half-life in both huFcRn transgenic mice and rhesus macaques. Due to their increased neutralizing potency and favorable biological and pharmacokinetic properties, FR3-loop-chimeric bNAbs are being considered for use in HIV prevention and treatment. 3) Structure-guided interdomain stabilization of the HIV-1 envelope trimer abrogates CD4 binding and improves immunogenicity. The inherent flexibility of the HIV-1 Env trimer and its ability to bind the CD4 receptor represent two of the major obstacles to the development of a vaccine capable of eliciting bNAbs. In particular, binding to CD4 not only induces conformational changes that rapidly compromise the native antigenic state of the trimer, but also occludes the CD4-binding site (CD4-BS), which is a critical antigenic target for antibody elicitation, and causes immunogen sequestration by CD4+ T cells, which lack antigen-presenting capabilities. To reduce the trimer flexibility and impair binding to CD4, we used a structure-guided approach to introduce neo-disulfide bonds bridging the inner and outer domains of gp120 (interdomain locks) in a region of molecular mimicry between gp120 and CD4 (SLWDQ). The neo-disulfide bridges effectively formed, as shown by mass-spectrometry analysis, and resulted in trimer stabilization in a native-like pre-fusion configuration. Of note, this design was successfully applied to both soluble trimers (SOSIP) and native full-length gp160 from different HIV-1 strains and clades. Interdomain-locked trimers showed increased thermal stability, reduced spontaneous misfolding, reduced or abrogated binding to non-neutralizing antibodies, enhanced binding to bNAbs and, most importantly, loss of CD4-binding activity. The crystal structure of an interdomain-stabilized trimer confirmed the formation of the expected disulfide bond and provided insights into the structural basis for CD4-binding impairment.